Location: Animal Diseases Research
Title: Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay Authors
Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 11, 2012
Publication Date: July 27, 2012
Repository URL: http://handle.nal.usda.gov/10113/56585
Citation: Madsen-Bouterse, S.A., Zhuang, D., Orourke, K.I., Schneider, D.A. 2012. Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay. Biochemical and Biophysical Research Communications. 423(4):770-774. Interpretive Summary: Classical scrapie in sheep and goats is a fatal brain disease caused by misfolded proteins called prions. Recent technical advances for research use have allowed for detection of prions in tissues and fluids from sheep where they were previously undetected by standard laboratory methods. Protein misfolding cyclic amplification, or PMCA, is one such technique that has yet to be extensively utilized in studies of scrapie in goats. We have applied this technique to brain tissue from scrapie infected goats and have shown that it can be used to successfully detect misfolded prion protein that was diluted past the point of detection by standard laboratory methods. PMCA requires a source of normally folded protein. Our results indicate that brain tissue from a transgenic mouse expressing the ovine prion protein can be used as the source of normally folded prion protein in the PMCA assay. Further, we show that in some cases PMCA, but not direct infection of transgenic mice, maintains the unique protein conformation of the original prion source. Further adaptation of this technique may lead to enhanced detection of prions in other tissues and bodily fluids and may help identify and discriminate various sources of scrapie prions in goats.
Technical Abstract: The protein misfolding cyclic amplification (PMCA) assay allows for detection of the disease associated isoform of the prion protein in tissues and fluids of sheep where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA. The aim of the current study was to assess the use of PMCA when evaluating scrapie derived from goats. Diluted brain homogenate from scrapie infected goats (i.e., the scrapie seed) was subjected to PMCA using normal brain homogenate from ovinized mice (tg338) as the source of normal cellular prion protein (PrP-C, the substrate). The end-point of the assay was detection of the proteinase K-resistant misfolded prion protein core (PrP-res) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analysis with and without sodium phosphotungstic acid precipitation demonstrated that PrP-res post-PMCA was more similar to the scrapie inoculum from goats than was PrP-res extracted from the brain of inoculated tg338. Thus, brain homogenate prepared from uninoculated ovinized mice serves as an appropriate substrate for PMCA of scrapie derived from goats. In addition, in vitro amplification with PrP-C from tg338 may provide some benefits over bioassay in tg338 in future analyses if it more faithfully maintains the misfolded conformation of PrP-res that accumulates during infection of the natural host in multiple scrapie strains.